Transcriptional Regulation of bcl-2 Mediated by the Sonic Hedgehog Signaling Pathway through gli-1

Rebecca L.H Bigelow,Nikhil S Chari,Anne Birgitte Undén,Kevin B Spurgers,Sangjun Lee,Dennis R Roop,Rune Toftgård,Timothy J Mcdonnell

doi:10.1074/jbc.m310589200

Abstract

Basal cell carcinomas (BCCs) express high levels of the antiapoptotic proto-oncogene, bcl-2, and we have shown that bcl-2 contributes to the malignant phenotype in a transgenic mouse model. The basis of bcl-2 transcriptional regulation in keratinocytes is unknown. The sonic hedgehog (SHH) signaling pathway is frequently altered in BCCs. Mediators of shh signaling include the downstream transactivator, gli-1, and transrepressor, gli-3. Seven candidate gli binding sites were identified in the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. Gli-1 was also able to up-regulate endogenous bcl-2. Gli-3 cotransfection resulted in no significant changes in bcl-2 promoter activity compared with control. Gli-3 has been demonstrated to be proteolytically processed into an N-terminal repressive form that can inhibit downstream transactivation by gli-1. Gli-3 mutants possessing only the N-terminal region or the C-terminal region were made and used in luciferase assays. The N terminus of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. Gel shift analysis and luciferase assays demonstrated that gli binding site 4 (-428 to -420), is important for gli transcriptional regulation. Skin samples from transgenic mice expressing an RU486 gli-1 transgene exhibited significantly higher levels of endogenous bcl-2 protein in epidermal keratinocytes as assessed by immunoblotting and immunohistochemistry. Together, these findings provide consistent evidence that gli proteins can transcriptionally regulate the bcl-2 promoter and that gli-3 can inhibit transactivation by gli-1. These studies further suggest that one consequence of the deregulation of shh signaling in BCC is the up-regulation of bcl-2.

Highlights

Basal cell carcinomas (BCCs) express high levels of the antiapoptotic proto-oncogene, bcl-2, and we have shown that bcl-2 contributes to the malignant phenotype in a transgenic mouse model
Loss of heterozygosity is frequently found at the location of patched, 9q22, in patients suffering from nevoid basal cell carcinoma syndrome who are predisposed to developing BCCs, and in sporadic BCC tumors [2, 15]
It has more recently been established that alterations in the shh signaling pathway occur in virtually all BCCs of the skin [2,3,4,5,6]

Summary

Introduction

Basal cell carcinomas (BCCs) express high levels of the antiapoptotic proto-oncogene, bcl-2, and we have shown that bcl-2 contributes to the malignant phenotype in a transgenic mouse model. Gel shift analysis and luciferase assays demonstrated that gli binding site 4 (؊428 to ؊420), is important for gli transcriptional regulation. Gel shift assays were done using 3 ␮g of cold translated protein incubated with probes corresponding to a positive control previously reported to bind to gli (ϩ) [35], a mutant oligonucleotide (Ϫ), and the seven probes corresponding to the potential gli binding sites in the bcl-2 promoter.

Results

Conclusion